Project Summary: Majority of infant leukemias involve rearrangements in the MLL gene and are most often fatal. There is a fundamental gap in our understanding of how MLL rearrangements cause leukemia. The long term goal is to advance the development of targeted therapies for MLL-fusion gene leukemia. The objective of this application is to identify the molecular pathways that mediate the pathogenesis of MLL-fusion gene leukemia. The experimental model studied will be knock-in mice carrying the MLL-AF4 or MLL-AF9 fusion gene. The first specific aim is to identify the downstream target genes of MLL-AF4 and MLL-AF9 that induce growth deregulation. The central hypothesis is that the key targets of the fusion proteins are the genes differentially expressed in the hematopoietic sub-population that displays the greatest growth deregulation. The experimental strategy will be comparison of gene expression profiles of purified hematopoietic progenitor/ stem cells from MLL-AF4, MLL-AF9 and wild type mice. Functional significance of the candidate genes will be tested using RNA interference in leukemia cell lines. The second specific aim is to identify mutations that co-operate with the MLL-fusion genes in the pathogenesis of leukemia. The knock-in mice display expansion of hematopoietic progenitors prenatally, yet leukemia development takes several months. The central hypothesis is that co-operating mutations will accelerate development of leukemia. Young mice will be infected with murine leukemia viruses that integrate into the genome and either activate or inactivate neighboring genes. Cloning and sequencing of integration sites will identify cooperating mutations. The cooperative actions of candidate genes will be tested by expressing them in knock-in bone marrow cells and transplanting these cells into recipient mice. The proposed studies are innovative since they address gene regulation by MLL-fusion proteins in a cell-specific context and will provide data on accessory mutational events in MLL-fusion gene leukemia. The proposed research is significant because it will enhance the understanding of the pathogenesis of MLL-rearranged leukemias. Relevance: Effective therapies for MLL-leukemias are limited by the lack of knowledge of the underlying biology of the disease process. The results of the proposed research will help the development of effective therapies for this dreadful disease.